Nucleotide sequence encoding for bifunctional enzyme for proline production

ABSTRACT

The invention is directed to the production of plants containing a cDNA clone encoding a bifunctional enzyme, delta 1  -pyrroline-5-carboxylate synthetase, hereinafter P5CS, with both gamma-glutamyl kinase and glutamic-gamma-semialdehyde dehydrogenase activities that catalyzes the first two steps in plant proline production, also called biosynthesis. The invention also provides methods for increasing the salt tolerance and drought resistance of plants, and for increasing the proline production and glutamine synthetase activity in plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No. 07/953,695 filed Sep. 29, 1992, now U.S. Pat. No. 5,344,923, issued Sep. 6, 1994.

FIELD OF INVENTION

1. Technical Field

The invention relates to (1) a novel bifunctional enzyme which catalyzes the first two steps in proline production in plants leading to osmoregulation in the plants, (2) a method to induce increased drought tolerance in a plant, (3) a method to induce increased sodium chloride tolerance in a plant, and (4) the plants produced thereby.

2. Background Art

Many plants synthesize and accumulate proline in response to osmotic stress. Drought and high salinity are the most important environmental factors that cause osmotic stress and impact negatively on plant growth and crop productivity. The role of proline biosynthesis in osmoregulation in bacteria is well established in, for example, Csonka, Microbiol. Rev., 53, 1989, pp. 121-147. The proline biosynthesis route in plants is not as well known but is thought to follow the biosynthesis route in bacteria. In Escherchia coli, the synthesis starts with the phosphorylation of glutamate by gamma-glutamyl kinase, hereinafter gamma-GK, which is encoded by the proB gene. The gamma-glutamyl phosphate thus formed is reduced to glutamic-gamma-semialdehyde, hereinafter GSA, by GSA dehydrogenase (GSD) which is encoded by the proA gene. The GSA thus produced spontaneously cyclizes to delta¹ -pyrroline-5-carboxylate (P5C) which is reduced by delta¹ -pyrroline-5-carboxylate reductase, hereinafter P5CR, which is encoded by the proC gene, to thereby form proline.

With the exception of P5CR which has been recently characterized (Krueger et al., Plant Physiol., 82, 1986, pp. 890-903; Rayapati et al., Plant Physiol., 91, 1989, pp. 581-586; Delauney and Verma, Mol. Gen. Genet., 221, 1990, pp. 299-305; Verbruggen et al., Abstract, Third International Congress, International Society for Plant Molecular Biology, Tucson, Ariz., Oct. 6-11, 1991), however, little is known about the other enzymes in this pathway in plants.

Therefore, it would be desirable to use genetic engineering of the proline production pathway in plants to counter osmotic stress to alter the level of a known osmoprotectant to thereby lead to a significant enhancement of crop performance under conditions of salt and drought stress.

SUMMARY OF INVENTION

The invention is directed to the isolation and identification of a mothbean (Vigna aconitifolia) cDNA clone encoding a bifunctional enzyme, delta¹ -pyrroline-5-carboxylate synthetase, hereinafter P5CS, with both gamma-glutamyl kinase and glutamic-gamma-semialdehyde dehydrogenase activities that catalyzes the first two steps in plant proline production, also called biosynthesis. By "bifunctional" herein is meant the ability of a single enzyme to perform two different catalyses and encode for gamma-GK and GSD.

The present invention is also directed to methods for increasing the sodium chloride tolerance and drought resistance of plants, such as but not limited to, tobacco, corn, wheat, barley, rye, spinach, lettuce, potatoes, carrots, navybeans, lima beans, soy beans, and the like, by incorporating therein the nucleotide sequence of SEQ ID NO: 1. "Plant" herein shall not be limited to the aforementioned crops, however, and shall include any and all vegetative materials.

The present invention was achieved by direct complementation of specific E. coli mutants with a Vigna nodule CDNA expression library. The isolated cDNAs have been fused to a strong plant promoter, and transferred to plants by Agrobacterium-mediated transformation. Transgenic Vigna roots have demonstrated overproduction of proline.

It is one object of the present invention to provide the complete nucleotide sequence of a P5CS CDNA clone, pVAB2.

Another object of the present invention is to provide a cDNA clone encoding a bifunctional enzyme, i.e., an enzyme that catalyzes both gamma-GK and GSD.

Yet another object of the present invention is to provide a method to overproduce proline and thus increase sodium chloride tolerance and drought resistance in a plant by the introduction into the plant of the P5CS CDNA clone encoding a bifunctional enzyme that catalyzes both gamma-GK and GSA production in said plant.

Yet another object of the present invention is to provide a method to increase glutamine synthetase (GS) activity in plants, thereby increasing the amount of substrate available for P5CS (glutamate) in the production of proline.

BRIEF DESCRIPTION OF THE FIGURES

In describing the preferred embodiment of the invention, which is illustrated in the Figures, specific terminology will be resorted to for the sake of clarity. However, it is not intended that the invention be limited to the specific terms so selected and it is to be understood that each specific term includes all technical equivalents which operate in a similar manner to accomplish a similar purpose. The words used to describe the invention are not literal limitations but include elements recognized as being equivalent by those skilled in the art.

FIGS. 1A, 1B, 1C and 1D are the complete nucleotide sequence of a P5CS cDNA clone, pVAB2, and primary sequence of the encoded V. aconitifolia delta¹ -pyrroline-5-carboxylate synthetase. The polypeptide region homologous to the E. coli gamma-GK (ProB) domain is underlined. The carboxy half of P5CS corresponds to GSA dehydrogenase (ProA). FIG. 1A and FIG. 1B show the first portion of the sequence and FIG. 1C and FIG. 1D show the latter portion of the sequence.

FIGS. 2A and 2B are a comparison of the V. aconitifolia P5CS amino acid sequence with A) the E. coli gamma-GK and B) GSA dehydrogenase sequences. Identical amino acids are indicated by a vertical line and similar amino acids are indicated by colons and periods. The conserved aspartic acid residues implicated in proline feedback inhibition are boxed, as are the leucine zipper sequences and the potential phosphorylation sites.

FIG. 3 is a graph illustrating feedback inhibition of delta¹ -pyrroline-5-carboxylate synthetase by proline. Ammonium sulfate-fractionated extracts from E. coli CSH26 (), E. coli CSH26 harboring PVAB2 (∘), the proB74 allele (∪), and the wild-type proB allele (□) were assayed for gamma-GK activity by the method of Hayzer and Leisinger, J. Gen. Microbiol., 118, 1980, pp. 287-293.

FIG. 4 illustrates the proline synthetic pathways in bacteria and plants, starting from glutamate. In bacteria, separate proB and proA steps are found for encoding gamma-GK and GSD, respectively, to produce GSA, whereas in plants, the glutamate is converted directly to GSA by the bifunctional enzyme, P5CS .

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention demonstrates the cause and effect relationship between proline levels in plants and osmotic stress. Since P5CR is not rate limiting in proline production, the P5CS must control the rate in this pathway. Therefore, P5CR CDNA was introduced in tobacco under the control of a constitutive promoter. Significant accumulation of proline was observed which was found to be directly correlated with water potential in transgenic plants. This indicates that such plants will be able to better resist drought and salinity conditions.

By the present invention, E. coli proB, proA, and proBA auxotrophic mutants were transformed by electroporation with a Vigna aconitifolia nodule cDNA expression library. A transformation efficiency of 10⁹ -10¹⁰ transformants/ug of DNA was routinely obtained by the present invention, which is about two orders of magnitude higher than that obtained by classical transformation methods. Thus electroporation significantly enhances the recovery of complemented mutants. One group of clones of the present invention was isolated by selection for growth of transformants on minimal media without proline. It has been determined by the present invention that these clones encode a novel bifunctional enzyme, P5CS, which has both gamma-GK and GSD activities. The P5CS clones produced according to the present invention efficiently complemented E. coli proB, proA, and proBA, but not proC mutants. The predicted amino acid sequence of the enzyme produced by the present invention revealed the presence of two domains with significant homology to the E. coli proA and proB proteins. The recombinant P5CS protein of the present invention demonstrated GSD-dependent gamma-GK enzyme activity.

The proline biosynthetic pathway gene encoding the bifunctional P5CS enzyme from mothbean was stably introduced and expressed in tobacco. The construct contained a CaMV-35S promoter, P5CS cDNA, and was introduced into tobacco using Agrobacterium--mediated transformation by techniques known to those skilled in the art. Plants that were transformed by the present invention were maintained on kanamycin. Southern blots were made with some of the genomic DNA transformants of the present invention and controls (wild type, also referred to herein as "WT" and pBI121, also referred to herein as "PBI") and were probed with Vigna P5CS cDNA. The results confirmed that 1 to 3 copies of P5CS gene were inserted. Ten independent primary transformants (F₀) that produced high proline were selected and self pollinated. The plants from F₁ generation were used for proline and amino acid determinations.

RNA and protein were extracted from leaves of several independent transformants and wild type plants, and northern and western blots were run using the protocols described hereinbelow. Blots showed the size and level of P5CS transcript (about 2.6 kb) after hybridization of the radioactively labeled Vigna P5CS cDNA probe to filters containing leaf RNA. To determine whether this mRNA is translated, western blots were run using polyclonal antibodies prepared against the mothbean P5CS enzyme. Total proteins from leaves were subjected to SDS-PAGE, and transferred to nitrocellulose membrane and probed with antibodies which revealed single band corresponding to mothbean P5CS. These data demonstrated that P5CS mothbean can be effectively expressed, and is stable, in, for example, tobacco.

Proline levels were found to be high in all transgenic plants of the present invention and the amounts ranged from about 830 to about 1590 mg/g fresh weight of leaves (from 10 individual lines), while the proline level ranged from 80 to 89 mg/g in control plants. These data indicate stable insertion and expression of P5CS gene in transgenic tobacco and the level of expression of P5CS was correlated with the level of proline production indicating that this enzyme is rate limiting in proline production.

The complete nucleotide sequence of a P5CS cDNA clone, pVAB2, is shown in FIGS. 1A, 1B, 1C and 1D. The 2417 bp sequence contains a single major open reading frame encoding a polypeptide of 73.2 kD, assuming that translation is initiated at the first ATG codon from the 5' end of the coding strand. Sequence comparison with the E. coli ProB and ProA proteins (FIGS. 2A and 2B) indicate that V. aconitifolia P5CS polypeptide has two distinct domains. The amino terminal domain of the P5CS protein showed 33.3% identity and 55.5% overall similarity to the E. coli ProB protein. A domain with 35.7% identity and 57.9% similarity to the ProA protein is located at the carboxyl end of the nucleotide sequence. An unexpectedly high level (42.4%) of the sequence similarity was found between the E. coli ProB and ProA proteins leading the inventor to the conclusion that the proB and proA genes may have arisen in the bacteria by duplication of an ancestral gene; the encoded proteins may have later acquired domains conferring the respective kinase and reductase activities of the present-day enzymes. It has been surprisingly discovered in the present invention that the corresponding plant genes have evidently fused to create the bifunctional enzyme identified herein from V. aconitifolia.

FIG. 4 illustrates the comparison between the proline production pathways in bacteria and plants and the genes involved. The key difference is the fusion or merging of the proB and proA catalytic steps of the bacteria into the single gene in the plant pathway to form GSA. Both the bacteria and the plant proline production pathways start with glutamate, then proceed through GSA to PSC which, in the presence of P5CR, is converted or reduced to proline. However, in bacteria, the glutamate is phosphorylated in the presence of gamma-GK encoded by proB to produce glutamyl phosphate (GP). The GP is then converted to GSA by GSD encoded by proA. In the plant, however, it has been discovered herein that the glutamate is converted directly to GSA by the bifunctional enzyme P5CS, shown in FIGS. 1A, 1B, 1C and 1D. The P5CS therefore has the combined properties of proB and proA in the proline synthetic pathway.

Interestingly, it has also been discovered herein that a "leucine zipper" sequence (see FIGS. 2A and 2B) is present in each of the enzymatic domains of Vigna P5CS. The leucine zippers mediate protein:protein dimerization in structural proteins of both eucaryotes and procaryotes as well as in transcriptional regulatory proteins. It is believed that the leucine zippers in P5CS function intramolecularly to maintain the proper tertiary structure of the two domains of this enzyme, and homodimer or heterodimer formation may occur through the leucine zippers to allow close association between the originally separate enzymes (proB and proA) fused in P5CS. It is also possible that these zippers are a relic of the time when the gamma-GK and GSA-dehydrogenase enzymes were separate and had to be brought together to form a functional complex, as in E. coli. Thus these leucine zippers may facilitate inter- and intra-molecular interactions. In addition, P5CS contains a potential phosphorylation site (FIGS. 2A and 2B).

P5CS has GSA dehydrogenase and gamma-GK activities, and the gamma-GK activity is feedback inhibited by proline. To confirm that the presently claimed sequence, PVAB2 of FIGS. 1A, 1B, 1C and 1D, encoded P5CS activity, ammonium sulfate-fractionated extracts of E. coli CSH26 and CSH26 harboring PVAB2 were assayed for GSA dehydrogenase-dependent gamma-GK enzyme activity. As shown by the essentially horizontal line () in FIG. 3, no gamma-GK activity was detected in E. coli strain CSH26, whereas CSH26 containing pVAB2 exhibited a high level of gamma-GK activity (see curve (∘)). Because proline production is regulated by end-product inhibition of gamma-GK in bacteria, the plant enzyme P5CS was similarly subjected to feed-back control testing. FIG. 3 shows that the Vigna enzyme is 50% inhibited by 6 mM proline. The wild-type E. coli gamma-GK (see curve (□) of FIG. 3) was 30 times more sensitive than the Vigna enzyme, i.e., it shows 50% inhibition at 0.2 mM proline, while a mutant form of the bacterial enzyme (encoded by the proB74 allele, curve (▪)) is approximately 200-fold less sensitive to end product inhibition. Thus, the sensitivity of the recombinant Vigna enzyme (curve (∘)) to feedback inhibition is much less than that of the wild-type E. coli enzyme (curve (□)) but more than that of the mutated gamma-GK enzyme. The mutation responsible for the resistance to end-product inhibition of the gamma-GK in the proB 74 allele involves a substitution of an A for a G nucleotide, resulting in a change from aspartic acid to asparagine residue at position 107. It is to be noted, however, that this aspartate residue is conserved in the Vigna P5CS enzyme of the present invention, raising the possibility that changing this residue to asparagine by site-directed mutagenesis may result in further diminution of feedback inhibition of the plant enzyme. In spite of the conserved aspartic acid residue implicated in feedback control, the Vigna enzyme is less sensitive to inhibition by proline than is the E. coli enzyme suggesting that other regions of the P5CS enzyme may also be important for feedback regulation.

By observing the sensitivity of the mothbean enzyme to end-product inhibition in proline synthesis, it has been discovered in the present invention that regulation of the pathway in plants is exerted primarily at the P5CS step.

To demonstrate the expression levels of the Vigna P5CS gene in different tissues and under conditions of osmotic stress, RNA from nodules, leaves, and roots, as well as roots from plants watered with 200 mM sodium chloride, was probed on a northern blot with the pVAB2 cDNA. The results showed that the P5CS transcript in Vigna leaves was more abundant than in the roots and nodules. The level of P5CS transcripts in roots was enhanced by treatment of the plant with 200 mM sodium chloride. An increase in the level of transcript of P5CS and P5CR during osmotic stress will therefore facilitate increased proline production from glutamate.

To demonstrate that P5CR activity is not a rate limiting step in proline production in plants, the soybean P5CR cDNA was placed under the control of cauliflower mosaic virus (CaMV-35S) promoter and introduced into Vigna via T-DNA mediated transformation. Regenerated plants showed 10 to 100 fold increase in the amount of P5CR activity in the leaves. Measurement of proline and P5C levels in the transgenic plants indicated that proline synthesis was limited by P5C production rather than the activity of P5CR. All P5C in control and transgenic plants was converted to proline. No significant accumulation of proline occurred in transgenic plants expressing high levels of P5CR because the specific activity of the P5CR of both stressed and unstressed Vigna cell cultures is hundreds of folds greater than the proline synthesis rate, and the enzyme of the present invention functions at only a small fraction of its V_(max). Therefore, P5CR is not rate limiting in the proline production pathway in plants.

Using the procedures described in Miao et al., The Plant Cell, Vol. 3, January 1991, pp. 11-12, the isolated cDNA of the present invention was transferred into transgenic Vigna plants to demonstrate the effect therein on proline production. Table I illustrates a comparison of control versus transgenic Vigna plants. The results illustrate that the proline content of the control plants (samples 1, 2 and 3) was low (0.76 to 2.42 micromoles of proline per gram of root tissue) relative to the proline content of the transgenic Vigna plants of the present invention (samples 4-8), i.e. 3.9 to 13.2 micromoles of proline per gram of root tissue. This represents a 10 to 100 fold increase in proline production in the transgenic plants as a result of the transference of the nucleotide sequence of the present invention.

                  TABLE 1                                                          ______________________________________                                         Concentrations of proline in control and                                       transgenic Vigna roots                                                         Sample  Tissue       ug proline/g                                                                               uM proline/g                                  Number  (hairy roots)                                                                               of fresh root                                                                              of fresh root                                 ______________________________________                                         1       Control (wt-1)                                                                              11          0.76                                          2       Control (wt-2)                                                                              22.2        1.54                                          3       Control (wt-3)                                                                              35          2.42                                          4       AH30-A7      160         11.08                                         5       AH30-10      190         13.2                                          6       AH30-Bl2     170         11.8                                          7       AH30-C8      56          3.9                                           8       AH30-55      130         9.0                                           ______________________________________                                    

Control tissue was obtained from roots transformed with pB1121 only.

Six week old seedlings of both transformed and control were subjected to 10-day water stress to see whether high proline helps in maintaining the leaf turgor. Proline levels were measured both before and after water stress. While proline level was approximately 14 folds higher before stress, it was only about 2 folds higher than controls after stress. Proline levels returned to the normal level upon rehydration indicating that a futile cycle is avoided. A gradient of proline concentration was observed with a declining trend from top leaves to the bottom. Control plants started wilting in 5-6 days, while wilting was delayed by at least two days in transgenic plants of the present invention and wilting was more severe in controls than in transgenic leaves. Measurements of water potential obtained from leaf disc and sap before stress were almost identical in control and high proline producing plants. However, the osmotic potentials of lower leaves (7th and 9th from top) in the controls were higher compared to the leaves at similar positions in the transgenic plants, as seen in Table 2.

                  TABLE 2                                                          ______________________________________                                         Osmotic Potential of Intact Leaf Discs                                         Before and After Drought Stress                                                      Before Stress (MPa)                                                                            After Stress (MPa)                                       Plant Leaf Number     Leaf Number                                              Line  3      5      7    9    3    5    7     9                                ______________________________________                                         WT    -2.34  -2.26  -2.49                                                                               -2.57                                                                               -3.93                                                                               -4.17                                                                               -4.66 Beyond                           PBI   -2.21  -2.53  -2.64                                                                               -2.59                                                                               -4.04                                                                               -4.89                                                                               Beyond                                                                               Beyond                           P5CS  -2.75  -2.66  -2.75                                                                               -2.58                                                                               -3.32                                                                               -3.58                                                                               -4.10 -4.62                            ______________________________________                                          Mean of 4 replicates each for WT and PBI.                                      Mean of 12 replicates for P5CS.                                          

The leaf sap of the WT, PBI and transformed inventive plants P5CS followed a similar trend, as seen in Table 3.

                  TABLE 3                                                          ______________________________________                                         Osmotic Potential of Leaf Sap                                                  Before and After Drought Stress                                                Osmotic Potential of Leaf Sap                                                  Before Stress (MPa)  After Stress (MPa)                                        Plant Line                                                                             Top     Middle  Lower  Top   Middle                                                                               Lower                               ______________________________________                                         WT      -0.78   -0.70   -0.74  -1.06 -1.11 -1.27                               PBI     -0.80   -0.74   -0.75  -0.98 -1.05 -1.26                               P5CS    -0.77   -0.77   -0.70  -0.70 -0.73 -0.73                               ______________________________________                                          Mean of 4 replicates of each from WT and PBI (controls) and mean of 12         replicates for P5CS. Leaf sap was squeezed from freshly picked leaves.   

Proline content was higher in leaves than in roots subjected to 500 mM NaCl.

As seen in Table 4, amino acid analysis of the plants of the present invention showed accumulation of proline after water stress, which increased at the expense of glutamate, precursor for proline.

                  TABLE 4                                                          ______________________________________                                         Effect of Water Stress on Total Amino Acids                                    in Control and Transgenic HPP Plants                                                      Before Stress                                                                              After Stress                                                       mol %       mol %                                                   Amino Acid   PBI    P5CS       PBI  P5CS                                       ______________________________________                                         Aspartate    24.99  11.96      4.72 1.71                                       Glutamate    27.57  12.83      3.48 7.63                                       Serine       3.77   2.13       1.43 0.97                                       Glycine      26.53  14.69      10.47                                                                               2.98                                       Histidine    0.00   0.65       0.00 0.16                                       Arginine     0.99   1.14       0.75 0.66                                       Threonine    3.26   1.95       1.41 1.27                                       Alanine      4.63   3.32       1.98 1.64                                       Proline      3.74   48.91      72.12                                                                               80.84                                      Tyrosine     1.61   0.70       0.55 0.19                                       Valine       1.15   0.55       1.66 0.43                                       Methionine   0.25   0.05       0.08 0.09                                       Cystine      nd     nd         nd   nd                                         Isoleucine   0.30   0.29       0.12 0.26                                       Leucine      0.59   0.33       0.14 0.38                                       Phenylalanine                                                                               0.35   0.28       0.59 0.38                                       Lysine       0.29   0.23       0.59 0.39                                       Tryptophan   nd     nd         nd   nd                                         ______________________________________                                    

This indicates that glutamate may act as the limiting factor for proline overproduction under water or salt stress. The total amount of proline in the control plants was 200 to 230 micrograms/gram fresh weight of leaves as opposed to 1120 micrograms/gram fresh weight in transgenic plants of the present invention. This illustrates that P5CS is the rate limiting factor in proline synthesis provided nitrogen is available. High levels of proline thus regulate P5CS activity.

Reduction in pod and seed formation were drastically affected in the control wild type (WT) plants when compared to transformants of the present invention under salt stress conditions. This reveals that proline can also protect the developmental traits. The root length and root dry weight per plant did not vary much between transgenic and control plants under unstressed conditions. However, significant differences were noticed for both root length and root dry weight per plant under stress, as shown in Table 5.

                  TABLE 5                                                          ______________________________________                                         Effect of Salinity Stress on Root Length                                       and Dry Weight in Transgenic Plants                                                   Root Length (cm)/plant                                                                       Root Dry Wt. (g)/plant                                    Plant Line                                                                              Unstressed                                                                               Stressed  Unstressed                                                                             Stressed                                  ______________________________________                                         WT       30.2(±0.56)                                                                           19.9(±1.22)                                                                           2.52(±0.04)                                                                         0.51(±0.02)                            PBI      30.6(±0.53)                                                                           19.1(±1.45)                                                                           2.70(±0.15)                                                                         0.55(±0.04)                            P5CS     33.3(±2.79)                                                                           27.3(±1.53)                                                                           2.69(±0.16)                                                                         1.25(±0.17)                            ______________________________________                                          Mean of 6 replicates with standard error (±) from control and               transgenic HPP plants.                                                   

Under progressive drought stress, many plants produce new lateral roots that do not form root hairs and accumulate proline. The present invention demonstrated the formation of such new roots and 5-fold more proline production (2-fold increase over old roots of the transgenic) only in transgenic plants that were grown in vermiculite, saturated with 400 mM NaCl. Roots that are exposed to salt or water stress lose their turgor and respond in an immediate cessation of elongation. Improved root growth in the transgenic plants of the present invention under salt stress conditions shows that the roots are better protected by proline accumulation. Therefore, development of such a new root system is an important component of the overall plant adaptation or strategy to cope with drought induced stress. The present invention thus demonstrates that proline enhances biomass production and facilitates flowering under stress conditions.

Salt stress significantly reduced the number of seeds formed per plant in the control plants compared to the transgenic plants of the present invention. Approximately 400 more seeds could be detected per pod in the transgenic plants than in the control plants, as shown in Table 6.

                  TABLE 6                                                          ______________________________________                                         Effect of Salinity Stress on Pod Formation                                     and Seed-Setting in Transgenic Plants                                                 No. of Pods/Plant                                                                            No. of Seeds/Pod                                          Plant Line                                                                              Unstressed                                                                               Stressed  Unstressed                                                                             Stressed                                  ______________________________________                                         WT       83.2(±5.62)                                                                           14.0(±1.27)                                                                           2641(±51)                                                                           965(±45)                               PBI      88.0(±2.16)                                                                           17.7(±1.29)                                                                           2664(±58)                                                                           1029(±48)                              P5CS     94.9(±1.67)                                                                           45.3(±1.41)                                                                           2516(±71)                                                                           1406(±29)                              ______________________________________                                          Plants were grown in metromix. Data represent mean of 4 to 5 replicates. 

    Effect of Ammonium Nitrate on Proline AccumulatioQn                                    Proline (μg/g fresh weight of leaves)                               Plant Line                                                                               Hoagland w/o nitrogen                                                                            20 mM NH.sub.4  NO.sub.3                           ______________________________________                                         WT        40.31(0.6)        230.59(3.6)                                        PBI       33.56(0.5)        200.70(1.8)                                        P5CS      629.05(40.3)      1120.23(64.2)                                      ______________________________________                                          Mean of 3 replicates each for WT and PBI.                                      Mean of 6 replicates from 2 independent transgenic lines for P5CS.       

It is known that salt and water stresses can severely affect the developmental traits such as flowering, fertilization and seed set. The age of the plant appears to be critical for the adaptation to saline stress. Plants that differentiated flower primordia (7 week old) before subjecting to stress (500 mM NaCl ) proceeded normally with flower blooming and pod formation in both transgenic and controls under stress. But, imposing the same stress to 6 week old plants (before the initiation of flowers) resulted in drastic reduction (average of 12 flowers as opposed to 80 in non-stressed plants) of flowering in transgenic while it was totally suppressed in the control plants. Experiments revealed that 300 Mm NaCl is critical for suppressing flower differentiation in control plants grown in vermiculite.

Thus, the invention provides a method of inducing both P5CS and P5CR genes by salt stress. The cloned plant gene of the present invention encodes the two enzymes in the plant which catalyze the first two reaction steps in the production of proline, long believed to provide a crucial role in osmoregulation. As demonstrated by the present invention, it is now possible to remove feedback control on proline production in plants and to produce the overexpression of proline from P5CS to confer salt and drought tolerance on a crop plant.

The invention further relates to increasing GS activity in plants thereby increasing the amount of substrate available for P5CS (glutamate) in the production of proline. This increase is achieved by introduction to the plant of the GS gene obtained by the procedures described in Miao et al., The Plant Cell, Vol. 3, January 1991, pp. 11-12. By this invention, the GS activity in the plant is increased relative to the level before introduction of the GS gene, whereby proline production is increased.

Experimental

Bacterial Growth Conditions E. coli mutant cells were grown at 37 degrees Centigrade in LB medium or in Minimal A medium (see Delauney and Verma, Plant Molecular Biology Manual, Kluwer Academic Publishers, Dordrecht, 1990, pp. A14: 1-22) supplemented with 0.2 Mm of required amino acids and 0.05 mM of thiamine. Ampicillin was added at 100 mg/l. The osmotic medium was increased by the addition of NaCl as indicated.

Plasmids and Bacterial Strains

Plasmids carrying wild-type proB (pDU1) and mutant proB74 (pDU101) allele were obtained from A. Dandekar (University of California, Davis). E. coli strains #X340, #W4032, #CSH26, and #X342 were obtained from B. Bachman, E. coli Genetics Stock Center, Yale University.

Construction of CDNA Expression Library

RNA was prepared from 21 day old mothbean (Vigna aconitifolia, L.) nodules by the method of Verwoerd et al., Nucl. Acid Res., 17, 1989, p. 2362, and poly(A)⁺ RNA was isolated by chromatography on an oligo(Dt)-cellulose column. Double stranded CDNA was synthesized, ligated to BstXI linkers, and size-fractionated on an agarose gel. Molecules greater than 500 bp in size were electroeluted from the gel, cloned into the BstXI-linearized pcDNAII plasmid vector (Invitrogen Corp., San Diego, Calif.), and E. coli DHIα cells were transformed. CDNA molecules inserted within the polylinkers of pcDNAII in the correct orientation and reading frame can be expressed under the control of the lacZ promoter.

Functional Complementation of E. coli Auxotrophs

E. coli proline auxotrophs were transformed by electroporation using ELECTRO CELL MANIPULATOR 600 apparatus (BTX, San Diego, Calif.) according to the manufacturers instructions. Briefly, cells were grown to late log phase in LB broth, pelleted by centrifugation, washed twice in ice-cold 10% glycerol in water solution, resuspended in cold 10% glycerol, and stored in 200 ul aliquots at -80° C. For electroporation, 20 ng of the pooled library DNA purified by double cesium chloride centrifugation was added to 200 ul of competent cells in a 2 mm cuvette and subjected to a 5 msec pulse at 2.5 kV/cm. The cells were immediately added to 10 ml of LB broth and incubated at 37° C. with shaking for 1 hour. The cells were then pelleted by centrifugation, washed with Minimal A medium, and plated into Minimal A plates containing ampicillin (100 ug/ml), IPTG (1 mM) and the amino acid supplements other than proline required by the respective mutants. To determine the transformation efficiencies, aliquots of electroporated cells were plated on the same media supplemented with proline (1 mM). Plated were incubated at 37° C. for two days. Plasmid DNA, prepared from single colonies grown in minimal media containing ampicillin, was used to retransform the mutants to confirm the complementation.

DNA Sequencing and Analysis

DNA sequencing was performed on CsCl-purified, double-stranded plasmid DNA by the dideoxynucleotide chain termination method using Sequenase II (U.S. Biochemicals, Cleveland, Ohio) according to the manufacturer's instructions. Deletions of the plasmid template were generated using Exonuclease III and mung-bean nuclease.

P5CS Enzyme Assay and Determination of End-product Feedback Inhibition

E. coli CSH26 cells and CSH26 harboring different plasmids were grown aerobically at 37° C. for about 16 hours. The cells were harvested by centrifugation at 4,000× g for 10 minutes, and the cell paste resulting was suspended in 4 ml of buffer A (50 mM Tris-Hcl, 1 mM dithiothreitol) at 40° C. Following sonication, (Benson Sonifier, Type 450) at 50 watts, the slurry was centrifuged at 4,000× g for 10 minutes and the supernatant fractionated by ammonium sulfate precipitation. For E. coli CSH26 containing PVAB2, the fraction precipitating between 0 and 30% saturation of ammonium sulfate was used for enzyme assay. The E. coli CSH26 and CSH26 harboring the proB74 or wild-type proB alleles, a fraction of 40 to 60% saturation was used. Ammonium sulfate precipitated fraction was dissolved in buffer A and dialyzed against the same buffer. Gamma-GK activity was assayed as a function of proline concentration (0.01 to 100 mM) to determine if proline synthesis is subject to end-product inhibition in plants.

Northern Blot Analysis

Poly(A)⁺ RNA from mothbean nodules, leaves and roots, as well as roots from plants watered with 200 mM NaCl, were electrophoresed in a 1.2% agarose-2.2M formaldehyde gel and transferred by vacuum blotting to a GENE SCREEN membrane (DuPont NEN, Boston, Mass.). The PVAB2 CDNA insert was labeled with ³² P-dCTP to a specific activity of 8×10⁸ dpm/ug using a random primer labeling system (Amersham, Arlington Heights, Ill.). Hybridization was performed by the method of Delauney and Verma, Mol. Gen. Genet., 221, 1990, pp. 299-305, and filters were exposed to x-ray film at -70° C.

While certain preferred embodiments of the present invention have been disclosed in detail, it is to be understood that various modifications may be adopted without departing from the spirit of the invention or scope of the following claims.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 1                                                   (2) INFORMATION FOR SEQ ID NO: 1:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2417 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double stranded                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (A) DESCRIPTION: Sequence encodes Pyrroline-5-carboxylate                      synthetase, a bifunctional enzyme consistimg of glutamyl                       kinase and glutamyl semialdehyde dehydrogenase activities                      (v) FRAGMENT TYPE: N-terminus: N-MESAVDPS--                                    C-terminus:-- RRRGWN-C                                                         Internal Fragment:--VDAREMAV--                                                 (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Vigna aconitifolia                                               (B) STRAIN: Mothbean                                                           (D) DEVELOPMENTAL STAGE: Root nodules                                          (vii) IMMEDIATE SOURCE:                                                        (A) LIBRARY: cDNA expression library                                           (B) CLONE: cDNA clone                                                          (x) PUBLICATION INFORMATION:                                                   (A) AUTHORS: Chien-An A. Hu, Ashton J. Delauney and                            Desh Pal S. Verma                                                              (B) TITLE: A bifunctional enzyme (delta1-pyrroline-5-                          carboxylate synthetase) catalyzes the first two                                steps in proline biosynthesis in plants                                        (C) JOURNAL: Proceeding of the National Academy of                             Science USA                                                                    (D) VOLUME: 89                                                                 (E) ISSUE: October                                                             (F) PAGES: 9354-9358                                                           (G) DATE: OCT-1992                                                             (K) RELEVANT RESIDUES IN SEQ ID NO: 1: FROM position 37 to                     position 2049 GenBank M92276                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                       TGCTCTAAAGGCTATTGCTCGTATCAGTGCTCAGCCATGGAGAGCGCG48                             MetGluSerAla                                                                   GTGGATCCTTCTCGGGGGTTCATGAAGGACGTGAAGCGTGTGATCATC96                             ValAspProSerArgGlyPheMetLysAspValLysArgValIleIle                               5101520                                                                        AAAGTTGGCACCGCGGTGGTCACTCGCGAAGAAGGAAGGTTAGCGGTT144                            LysValGlyThrAlaValValThrArgGluGluGlyArgLeuAlaVal                               253035                                                                         GGAAGATTGGGAGCTCTGTGCGAGCAGATTAAGCAACTCAACTCTCTC192                            GlyArgLeuGlyAlaLeuCysGluGlnIleLysGlnLeuAsnSerLeu                               404550                                                                         GGATACGACATTATACTCGTCTCCTCTGGCCCCGTCGGTATTGGACGC240                            GlyTyrAspIleIleLeuValSerSerGlyProValGlyIleGlyArg                               556065                                                                         CAAAGGCTACGTTTCCGTAAATTAATCAACAGCAGCTTCGCCGACCTT288                            GlnArgLeuArgPheArgLysLeuIleAsnSerSerPheAlaAspLeu                               707580                                                                         CAGAAACCCCAACTCGAACTCGACGGCAAGGCCTGCGCCGCCGTTGGA336                            GlnLysProGlnLeuGluLeuAspGlyLysAlaCysAlaAlaValGly                               859095100                                                                      CAGAACAGTCTCATGGCTCTCTACGATACGCTGTTCACTCAGCTCGAT384                            GlnAsnSerLeuMetAlaLeuTyrAspThrLeuPheThrGlnLeuAsp                               105110115                                                                      GTGACATCGGCTCAGCTTCTTGTGACGGATAACGATTTTCGAGATAAG432                            ValThrSerAlaGlnLeuLeuValThrAspAsnAspPheArgAspLys                               120125130                                                                      GATTTCAGGAAGCAGCTTACTGAGACTGTGAAGTCGCTGTTGGCGCTG480                            AspPheArgLysGlnLeuThrGluThrValLysSerLeuLeuAlaLeu                               135140145                                                                      AAGGTTATTCCGGTGTTCAATGAGAACGATGCCGTTAGTACCAGGAAG528                            LysValIleProValPheAsnGluAsnAspAlaValSerThrArgLys                               150155160                                                                      GCTCCCTATGAGGATTCTTCTGGTATATTTTGGGATAATGATAGTTTA576                            AlaProTyrGluAspSerSerGlyIlePheTrpAspAsnAspSerLeu                               165170175180                                                                   TCTGCTTTATTAGCCTTGGAGTTAAAAGCCGATCTCCTTGTTTTGTTG624                            SerAlaLeuLeuAlaLeuGluLeuLysAlaAspLeuLeuValLeuLeu                               185190195                                                                      AGTGATGTAGAAGGTCTTTACAGTGGCCCTCCAAGTGACCCTCATTCA672                            SerAspValGluGlyLeuTyrSerGlyProProSerAspProHisSer                               200205210                                                                      AAGCTTATTTATACATATAACAAAGAAAAACATCAGAATGAAATTACT720                            LysLeuIleTyrThrTyrAsnLysGluLysHisGlnAsnGluIleThr                               215220225                                                                      TTTGGCGACAAGTCTAGAGTGGGAAGAGGCGGAATGACTGCCAAAGTA768                            PheGlyAspLysSerArgValGlyArgGlyGlyMetThrAlaLysVal                               230235240                                                                      AAAGCTGCGGTTCATGCAGCTGAAGCTGGCATTCCTGTTGTTATTACC816                            LysAlaAlaValHisAlaAlaGluAlaGlyIleProValValIleThr                               245250255260                                                                   AGTGGTTTTGCACCTGAGAATATCATTAATGTTCTCCAAGGACAACGT864                            SerGlyPheAlaProGluAsnIleIleAsnValLeuGlnGlyGlnArg                               265270275                                                                      ATAGGAACTCTCTTCCATAAAGATGCACATGAGTGGGCTCAAGTAAAA912                            IleGlyThrLeuPheHisLysAspAlaHisGluTrpAlaGlnValLys                               280285290                                                                      GAGGTTGATGCACGTGAGATGGCTGTTGCAGCAGGGAATGTTCGAGAA960                            GluValAspAlaArgGluMetAlaValAlaAlaGlyAsnValArgGlu                               295300305                                                                      GGCTCCAGGCGTTATCTTCAGAGGAAAGGAAACAAAATTTTACTTAAA1008                           GlySerArgArgTyrLeuGlnArgLysGlyAsnLysIleLeuLeuLys                               310315320                                                                      ATAGCTGATGCCCTGGAAGCAAATGAAAAAATAATCAGGATTGAAAAT1056                           IleAlaAspAlaLeuGluAlaAsnGluLysIleIleArgIleGluAsn                               325330335                                                                      GAAGCTGATGTTACTGCTGCACAAGAAGCAGGATATGAAAAATCCTTG1104                           GluAlaAspValThrAlaAlaGlnGluAlaGlyTyrGluLysSerLeu                               345350355                                                                      GTGGCTAGGCTAGCTTTAAAACCTGGGAAGATTGCAAGTCTTGCAAAC1152                           ValAlaArgLeuAlaLeuLysProGlyLysIleAlaSerLeuAlaAsn                               360365370                                                                      AACATGCGAATCATTGCCAATATGGAAGATCCAATTGGTCGAGTATTA1200                           AsnMetArgIleIleAlaAsnMetGluAspProIleGlyArgValLeu                               375380385                                                                      AAACGTACCGAGCTTTCAGATGGGCTAATTTTAGAAAAGACATCATCT1248                           LysArgThrGluLeuSerAspGlyLeuIleLeuGluLysThrSerSer                               390395400                                                                      CCTTTGGGAGTGCTCCTTATTGTTTTTGAGTCACGTCCTGATGCTCTT1296                           ProLeuGlyValLeuLeuIleValPheGluSerArgProAspAlaLeu                               405410415420                                                                   GTACAGATAGCTTCATTGGCAATCCGAAGTGGGAATGGGCTTCTCTTG1344                           ValGlnIleAlaSerLeuAlaIleArgSerGlyAsnGlyLeuLeuLeu                               425430435                                                                      AAAGGTGGCAAAGAAGCTAAGCGATCAAATGCAATTTTGCACAAAGTA1392                           LysGlyGlyLysGluAlaLysArgSerAsnAlaIleLeuHisLysVal                               440445450                                                                      ATTATCGAGGCCATACCAGATAATGTTGGTGGAAAACTTATAGGACTT1440                           IleIleGluAlaIleProAspAsnValGlyGlyLysLeuIleGlyLeu                               455460465                                                                      GTGACCTCAAGGGAAGAGATCCCTGAGCTACTTAAGTTGGATGATGTA1488                           ValThrSerArgGluGluIleProGluLeuLeuLysLeuAspAspVal                               470475480                                                                      ATTGATCTGGTAATTCCAAGAGGCAGTAACAAACTTGTTTCTCAGATC1536                           IleAspLeuValIleProArgGlySerAsnLysLeuValSerGlnIle                               485490495500                                                                   AAGAGTTCAACTAAAATTCCTGTTTTAGGTCATGCTGATGGAATTTGC1584                           LysSerSerThrLysIleProValLeuGlyHisAlaAspGlyIleCys                               505510515                                                                      CATGTCTATGTTGATAAGTCTGCTAACGTGGAGATGGCAAAGCGGATT1632                           HisValTyrValAspLysSerAlaAsnValGluMetAlaLysArgIle                               520525530                                                                      GTATTAGATGCAAAAGTTGATTATCCGGCAGCCTGCAATGCCATGGAA1680                           ValLeuAspAlaLysValAspTyrProAlaAlaCysAsnAlaMetGlu                               535540545                                                                      ACACTTCTTATCCACAAGGATTTGATAGAGAAAGGTTGGCTTAAGGAG1728                           ThrLeuLeuIleHisLysAspLeuIleGluLysGlyTrpLeuLysGlu                               550555560                                                                      ATCATTCTTGACCTTCGAACTGAAGGCGTTATATTATATGGTGGCCCT1776                           IleIleLeuAspLeuArgThrGluGlyValIleLeuTyrGlyGlyPro                               565570575580                                                                   GTGGCAAGTTCTCTGTTAAATATTCCACAAGCACATTCATTTCATCAT1824                           ValAlaSerSerLeuLeuAsnIleProGlnAlaHisSerPheHisHis                               585590595                                                                      GAGTACAGTTCGCTGGCTTGCACCGCCGAAATTGTGGATGACGTGTAT1872                           GluTyrSerSerLeuAlaCysThrAlaGluIleValAspAspValTyr                               600605610                                                                      GCAGCTATTGATCATATAAATCTGTATGGAAGTGCACATACTGATTCG1920                           AlaAlaIleAspHisIleAsnLeuTyrGlySerAlaHisThrAspSer                               615620625                                                                      ATCGTTGCTGAAGATAACGAAGTAGCTAATGTGTTTCTACGCCAAGTA1968                           IleValAlaGluAspAsnGluValAlaAsnValPheLeuArgGlnVal                               630635640                                                                      GACAGTGCTGCTGTTTTTCACAATGCAAGCACCAGATTCAGTGATGGG2016                           AspSerAlaAlaValPheHisAsnAlaSerThrArgPheSerAspGly                               645650655660                                                                   GCACGATTTGAGACTAGGCGCAGAGGTTGGAATTAGTACAAGC2059                                AlaArgPheGluThrArgArgArgGlyTrpAsn                                              665670                                                                         AGGATTCATGCTCGAGGTCAGTAGGAGTTGAGGATTGTTAACAACAAGAT2109                         GGATACTAAAAGGAAGGGACAAGTGGTAGATGGTGATAGAGGCGTTGTCT2159                         ACACCCACAAAGACCTTGCAATTTAATTTTAATGGTGCTTTGATTCCTTT2209                         TGTAGCCTTTTCGTTTGTTTTTTTTTTTTCACAGTAGAGAACGGCATTTG2259                         TACGGTTAAATAAAACCGGGTAATTTATGTCATTATTTGCTTGTTCCTTT2309                         TTGTCTAGAATCTTTACTGTCAACAATTATGGTCCACAATGTTTAACGAT2359                         TCTTGAATGACTACAATTTCAATTTGAGTTAATTTTTATATGTAAAAAAA2409                         AAAAAAAA2417                                                                   __________________________________________________________________________ 

We claim:
 1. A method to induce increased sodium chloride tolerance in a plant by the introduction into the plant of the P5CS cDNA clone encoding a bifunctional enzyme that catalyzes both gamma-glutamyl phosphate and glutamic-gamma-semialdehyde production in said plant.
 2. A method to induce increased drought resistance in a plant by the introduction into the plant of the P5CS cDNA clone, pVAB2, encoding a bifunctional enzyme that catalyzes both gamma-glutamyl phosphate and glutamic-gamma-semialdehyde production in said plant.
 3. A method to increase the production of proline in a plant comprising introducing into the plant the P5CS cDNA clone, pVAB2, shown in SEQ ID NO:
 1. 4. A plant with increased sodium chloride tolerance induced by the method of claim
 1. 5. A plant with increased drought resistance induced by the method of claim
 2. 6. A plant with increased proline production induced by the method of claim
 3. 7. A method according to claim 1 further comprising transforming the plant to express a glutamine synthetase gene to increase the amount of glutamate available as a substrate for the P5CS introduced to increase proline production.
 8. A plant with increased glutamine synthetase activity induced by the method of claim
 7. 9. The method of claim 1 wherein the plant is selected from the group consisting of tobacco, corn, wheat, barley, rye, spinach, lettuce, potatoes, carrots, navy beans, lima beans and soy beans.
 10. The method of claim 2 wherein the plant is selected from the group consisting of tobacco, corn, wheat, barley, rye, spinach, lettuce, potatoes, carrots, navy beans, lima beans and soy beans.
 11. The method of claim 3 wherein the plant is selected from the group consisting of tobacco, corn, wheat, barley, rye, spinach, lettuce, potatoes, carrots, navy beans, lima beans and soy beans.
 12. The method of claim 7 wherein the plant is selected from the group consisting of tobacco, corn, wheat, barley, rye, spinach, lettuce, potatoes, carrots, navy beans, lima beans and soy beans.
 13. The plant of claim 4 wherein the plant is selected from the group consisting of tobacco, corn, wheat, barley, rye, spinach, lettuce, potatoes, carrots, navy beans, lima beans and soy beans.
 14. The plant of claim 5 wherein the plant is selected from the group consisting of tobacco, corn, wheat, barley, rye, spinach, lettuce, potatoes, carrots, navy beans, lima beans, and soy beans.
 15. The plant of claim 6 wherein the plant is selected from the group consisting of tobacco, corn, wheat, barley, rye, spinach, lettuce, potatoes, carrots, navy beans, lima beans and soy beans.
 16. The plant of claim 8 wherein the plant is selected from the group consisting of tobacco, corn, wheat, barley, rye, spinach, lettuce, potatoes, carrots, navy beans, lima beans, and soy beans. 